Cytoplasmic forces functionally reorganize nuclear condensates in oocytes

Cells remodel their cytoplasm with force-generating cytoskeletal motors. Their activity generates random forces that stir the cytoplasm, agitating and displacing membrane-bound organelles like the nucleus in somatic and germ cells. These forces are transmitted inside the nucleus, yet their consequences on liquid-like biomolecular condensates residing in the nucleus remain unexplored. Here, we probe experimentally and computationally diverse nuclear condensates, that include nuclear speckles, Cajal bodies, and nucleoli, during cytoplasmic remodeling of female germ cells named oocytes. We discover that growing mammalian oocytes deploy cytoplasmic forces to timely impose multiscale reorganization of nuclear condensates for the success of meiotic divisions. These cytoplasmic forces accelerate nuclear condensate collision-coalescence and molecular kinetics within condensates. Disrupting the forces decelerates nuclear condensate reorganization on both scales, which correlates with compromised condensate-associated mRNA processing and hindered oocyte divisions that drive female fertility. We establish that cytoplasmic forces can reorganize nuclear condensates in an evolutionary conserved fashion in insects. Our work implies that cells evolved a mechanism, based on cytoplasmic force tuning, to functionally regulate a broad range of nuclear condensates across scales. This finding opens new perspectives when studying condensate-associated pathologies like cancer, neurodegeneration and viral infections.

| Immunocytochemistry screen of diverse nuclear biomolecular condensates in growing oocytes with classification into chromatin-associated and chromatin-free (droplet) condensates (a) Representative immunostainings of screened nuclear biomolecular condensates using known immunomarkers 7,8 relative to oocyte growth stages; NPAT (Nuclear Protein, Coactivator of Histone Transcription) is a marker of Histone Locus Bodies, PSPC1 (Paraspeckle Component 1) of paraspeckles, SMN1 (Survival of Motor Neuron 1) of gems and rarely associated with Cajal bodies, Fibrillarin of nucleoli and rarely Cajal bodies, TDP-43 (TARDBP; TAR DNA-Binding Protein 43) of paraspeckles, of TDP-43 bodies and of Cajal bodies, Coilin of Cajal bodies, and nuclear speckles stained with Abcam ab11826 antibody (SC35/SRSF2); Fibrillarin is notably nucleolar in NSN cells before becoming mostly nucleoplasmic as of the Trans stage suggesting the restructuring of the multiphase nucleolus 9 ; due to this nucleolar phase restructuring observed as of the Trans stage, the nucleolus (marked with the letter "n") loses Fibrillarin positivity and becomes recognizable only by signal exclusion and the surrounding DNA (stained with DAPI); asterisk in the SMN1 row indicates a visible nucleolar fusion; single 0.5μm z-planes are shown except for nuclear speckles which are 20μm z-projections; nucleus regions are outlined with dashed lines color coded relative to cytoplasmic stirring intensities at different growth stages. (b) Proportions of NSN, Trans, and SN oocytes that contain tested nuclear biomolecular condensates in their nucleus; cell number, NPAT NSN=14, Trans=6, SN=13, PSPC1 NSN=11, Trans=8, SN=26, SMN1 NSN=16, Trans=9, SN=24, Fibrillarin NSN=19, Trans=11, SN=14, TDP-43 NSN=9, Trans=10, SN=22, Coilin NSN=24, Trans=22, SN=56, nuclear speckles NSN=37, Trans=24, SN=42. (c) Images from the immunocytochemistry screen showing the two subpopulations of nuclear biomolecular condensates observed at various stages of oocyte growth; smaller, irregularly shaped chromatin-associated condensates indicated by fuchsia arrowheads and larger, round chromatin-free droplet condensates indicated by orange arrowheads; DNA is stained with DAPI; all images are z-projections, 3μm for NPAT and PSPC1, 2μm for SMN1, 5μm for Fibrillarin and bottom SRSF2, 10μm for TDP-43 and Coilin, and 1μm for top nuclear speckles; note that in the bottom nuclear speckles merge, a Coilin droplet is shown in yellow; note also that the nucleolus is an exception to the binary rule of classification since it always is a droplet that is in tight contact with and surrounded by chromatin. (d) Proportions of NSN, Trans, and SN oocytes containing nuclear condensates that are exclusively chromatin-associated (purple) or containing both chromatin-associated condensates and droplets (yellow); tested condensate immunomarkers and growth stages are indicated below the histogram; cell number, NPAT NSN=8, Trans=3, SN=1, PSPC1 NSN=9, Trans=8, SN=9, SMN1 NSN=0, Trans=1, SN=16, Fibrillarin NSN=17, Trans=11, SN=14, TDP-43 NSN=5, Trans=20, SN=22, Coilin NSN=11, Trans=21, SN=56, nuclear speckles NSN=37, Trans=24, SN=42. Bars above images are color coded relative to cytoplasmic stirring intensities at corresponding growth stages; scale bars, 5μm.Source data are provided as a Source Data file.

Movie 1 | Live-imaging of cytoplasmic random stirring in growing Control and fully-grown Control or F-actin mutant oocytes
Time-lapse stream-mode imaging in bright-field of a Control growing (NSN from a mid-antral follicle; left) oocyte and fully-grown Control and FMN2 -/mouse oocytes (SN; center and right) showing the increase in short-timescale cytoplasmic stirring with growth and disrupted activity in the fully-grown Factin mutant; note the peripheral nuclei in Control NSN and FMN2 -/cells and the presence of 2 visible nucleoli in the NSN nucleus. See Fig.1 and Supplementary Fig.1. Time in mm:ss; scale bar, 5µm.

Movie 2 | Live-imaging of SRSF2-GFP droplet coalescence in the fully-grown oocyte nucleus
Time-lapse stream-mode imaging of two nucleoplasmic SRSF2-GFP droplets fusing in an SN oocyte; SRSF2 is a nuclear speckle marker. See Supplementary Fig.3. Time in mm:ss; scale bar, 5µm.

Movie 3 | Live-imaging of local nuclear SRSF2-GFP droplet displacements and collisioncoalescence driven by cytoplasmic stirring in growing oocytes
Time-lapse stream-mode imaging of nucleoplasmic SRSF2-GFP droplets physically pushed by cytoplasm-based random kicks of the nuclear membrane in NSN and SN oocytes; droplet collisioncoalescence is instigated by the cytoplasm-based forces that locally displace the NSN droplet; note the more rapid diffusive dynamics of the SN droplet. See Fig.2 and Supplementary Fig.5. Time in mm:ss; scale bar, 5µm.

Movie 5 | Live-imaging of nuclear SRSF2-GFP droplets with random large-scale displacements and collision-coalescence in growing oocytes
Time-lapse images (50 µm z-projections) of global nucleoplasmic SRSF2-GFP droplet dynamics in Trans and SN oocytes on longer timescales; note random large-scale displacements of droplets and occasional collision-coalescence. See Fig.2 and Supplementary Fig.6. Time in hh:mm; scale bar, 5µm.
Movie 7 | 3D-simulations of nuclear SRSF2 droplet collision-coalescence evolution from NSN-like to SN-like states relative to cytoplasmic force intensity Time-lapse images of 3D-simulations on hour to day timescales showing the evolution of nuclear SRSF2-like droplet collision-coalescence relative to varying intensities of cytoplasmic stirring activity;